**3. Discussion**

Shake-flask batch fermentations were carried out to investigate the physiological behavior of the yeas<sup>t</sup> *Y. lipolytica* ACA-YC 5031 cultivated on biodiesel-derived glycerol employed as the sole carbon source in nitrogen-limited media enriched with OMWs, into which different NaCl quantities were added. The ability of the yeas<sup>t</sup> to convert crude glycerol into value-added metabolic products

was examined, as well as its potential to remove phenolic compounds and color from the medium. The obtained results seemed promising since they demonstrated the ability of the yeas<sup>t</sup> to produce value-added compounds under all circumstances, whereas simultaneously remarkable detoxification (decolorization and removal of phenolic compounds) of the employed wastewaters was realized. The amount of the produced secondary metabolites, however, varied significantly between the trials performed, since the addition of OMWs and NaCl (in various concentrations) had serious impact upon the physiology of the studied strain. The yeas<sup>t</sup> performed a remarkable growth ( *X* = 10.8 g/L, *YX*/*Glol* ≈ 0.12–0.15 g/g) in a nitrogen-limited glycerol-based media (*Glol0* ≈ 70.0 g/L) comparable to previous studies [20,35,40]. However, in other cases in which trials of *Y. lipolytica* or other yeasts of *Yarrowia* clade (i.e. *Y. bubula*, *Y. phangngensis*, etc) cultivated in shake-flasks or bioreactor experiments were carried out, significantly higher DCW quantities (i.e. *X* > 20.0 g/L, in some cases *X* ≈ 40.0 g/L) were recorded [54–57].

The presence of OMW (added in initial phenolic compounds ≈2.0 g/L, a typical concentration of phenolic compounds found in OMWs liberated from 3.0- and 2.5-phase centrifuge operation modules [58]) slightly inhibited the cell growth, reducing the DCW to 8.7 g/<sup>L</sup> as previously reported due to the high toxic e ffect of the e ffluent [3,4,58]. The simultaneous addition of increased salt concentrations resulted in further growth reduction, while the produced intra-cellular polysaccharides did not reach very high values. This phenomenon was reasonable and expected, taking into account that conditions of osmotic stress do not induce cell growth, in contrast to citric acid and polyols production.

Sarris et al. [3] reported a small biomass reduction of related *Y. lipolytica* strains after the addition of 2.0 g/<sup>L</sup> phenolic compounds in a medium containing 35.0 g/<sup>L</sup> glucose, the principal sugar found in OMWs, and a compound that is metabolized following similar metabolic pathways as those of glycerol. The produced citric acid (=18 g/L) and the conversion yield on glucose consumed (≈0.70 g/g) were not a ffected and remained almost constant. Similarly, Sarris et al. [4] reported a slightly reduced growth of the strain *Y. lipolytica* ACA-DC 5029 after the addition of OMW in a glycerol-based medium. In that study, the addition of OMW significantly increased citric acid production from 10.5 g/<sup>L</sup> (*YCit*/*Glol* = 0.15 g/g) to 32.7 g/<sup>L</sup> ( *YCit*/*Glol* = 0.52 g/g). These results, accompanied with previous studies that reported the successful usage of OMWs in order to dilute concentrated glycerol-based media, sugges<sup>t</sup> that these wastewaters are a suitable medium for citric acid production [4,58]. In the present investigation, it was also demonstrated that addition of salt, combined with presence of OMW in the culture medium at ideal pH conditions (pH = 5.0–6.0), resulted in biosynthesis of citric acid by *Y. lipolytica* at remarkably high quantities. The addition of phenols and the increased concentrations of NaCl significantly favored the production of citric acid, which it was steadily increased, reaching a maximum quantity of 54.0 g/<sup>L</sup> and a *YCit*/*Glol* value = 0.82 g/g. On the other hand, Tomaszewska et al. [59] observed decreased citric acid production in low pH conditions (pH = 3.0), while the polyols were significantly increased. In contrast, Rzechonek et al. [60] reported the ability of a genetically modified *Y. lipolytica* to produce a high amount of citric acid, up to 75.8 g/L, on media containing crude glycerol at pH = 3.0. E fficient citric acid production by *Y. lipolytica* strains occurs normally in nitrogen-limited glycerol-based media and pH values between 5.5 and 7.0 [38,40,59]. The results achieved in the current study (*Citmax* = 54.0 g/<sup>L</sup> with *YCit*/*Glol* = 0.82 g/g) compares favorably with the ones obtained in shake-flask and batch-bioreactor experiments for both the absolute (g/L) and relative (g per g of glycerol consumed) values of produced citric acid. Nevertheless, higher citric acid levels have been reported for conversions carried out in fed-batch bioreactors. A summary of the findings for the conversion of crude glycerol and olive-mill wastewater–based media to citric acid by *Y. lipolytica* strains in various fermentation configurations, including the current study, is given in Table 4.

Erythritol and mannitol were the main byproducts of glycerol metabolism. It is worth noting that traces of arabitol were also observed in the culture medium with and without OMW. However, production of arabitol was not observed in salt containing fermentations, indicating that both the salt and the citric acid inhibited its production, whereas mannitol and erythritol were synthesized at low concentrations. More specifically, mannitol and erythritol production reached maximum values 13.4 g/<sup>L</sup> and 8.8 g/L, respectively, in the glycerol-based medium, while addition of OMW reduced their production. Rymowicz et al. [26] and Chatzifragkou et al. [42] reported that it is essential for polyol biosynthesis an appropriate high initial glycerol concentration. Although high glycerol concentration favored the production of mannitol, Rywi ´nska et al. [34] reported higher erythritol production than mannitol in a crude glycerol substrate. Moreover, it has been found that growth of osmophilic yeasts in glycerol-based medium containing salt at low pH values promoted erythritol production [23,39,40], a fact that was not validated in the current investigation since, as it has been previously reported, the addition of salt in OMW/glycerol blends clearly shifted the cellular metabolism towards citric acid production instead of polyols (see Figure 2a–e).


**Table 4.** Metrics of citric acid production from glycerol- or OMW-based media by *Yarrowia lipolytica* strains cultivated on various fermentation configurations.

\* »: same as above; a: *D* = 0.009 h−1; b: *D* = 0.021 h−1; c: Trial under non-aseptic conditions; d: Total citric acid, sum of citric and iso-citric acid;

Although the biochemical events leading to the synthesis of polyols from glycerol in *Y. lipolytica* strains have not been completely elucidated [20,35,42], it appears that the biosynthesis of polyols depends on the response of the enzyme complexes to the external osmotic environment. More specifically, high osmotic pressure increases the activity of the enzyme erythrose reductase (EP), while it reduces the activity of the enzyme mannitol-1-phosphate dehydrogenase (M-1-PDH) [23]. In

this study, under all circumstances where salt was added, mannitol production was observed after 24 h incubation until the end of the fermentations, while erythritol production was observed later and for a very short period. Since erythritol molecule with a smaller MW than the mannitol, the osmotic pressure that comes from erythritol is higher than that of mannitol at the same concentrations of both compounds. When the microorganism is exposed to a high osmotic pressure, it produces erythritol to balance the osmotic pressure extra-cellularly and intra-cellularly. The organism accumulates a high amount of erythritol, which compensates for the di fference between the extracellular and intracellular water potential [27]. Other parameters that a ffect erythritol production are the pH of the culture medium (production of polyols is mainly performed at pH ranging between 2.5 and 3.5), the incubation temperature, the type and the concentration of the carbon source employed, the nitrogen and phosphate source, as well as the presence of some metals [59]. On the other hand, many studies have reported mannitol to be the only polyol produced by the yeas<sup>t</sup> in order to be protected against the osmotic stress. Without doubt, the cooperative activity between those two polyols can protect the cell from damages by that kind of stress [23]. In general, the production of polyols from glycerol is not common for *Y. lipolytica* strains and can be very interesting for Food Technology.

*Y. lipolytica* is a well-known lipid-producing microorganism employed in the literature for the production of common and non-common fatty acids due to its ability to accumulate over 20.0% and up to 70.0% lipids per dry weight ( *w*/*w*) [51,64]. Dobrowolski et al. [65] have reported lipid content 25% of DCW in *Y. lipolytica* A101 on media containing crude glycerol from soap production, similar to this study on the glycerol-based medium. Lipid production by *Y. lipolytica* ACA YC-5031 increased from 23.9% of DCW on glycerol-based medium to 28.3% after the addition of phenolic compounds into the glycerol-based medium. Similarly, Sarris et al. [3,4] reported a proportionally increased in SCO production after the addition of OMWs in the culture medium. These results confirm the potential usage of OMW as a "lipogenic" substrate [3,58]. The addition of salt combined with OMW in the culture medium favored the production of intra-cellular lipids, which increased with increased salt concentration, reaching *YL*/*Xmax* = 35.1% *w*/*w* when 5.0% *w*/*v* NaCl had been added.

Biosynthesis of both citric acid and SCO from carbon sources like glycerol or similarly metabolized compounds in *Y. lipolytica* are processes that present remarkable similarities in their first steps being triggered by the depletion of an essential nutriment, and in particular nitrogen, from the culture medium [37,51,61,64,66–68]. Upon nitrogen limitation and in the presence of excess carbon, *Y. lipolytica* yeas<sup>t</sup> strains produce large amounts of TCA cycle intermediates, including citric acid and iso-citric acid, which are not further catabolized. In essence, nitrogen exhaustion results in a rapid decrease of the intra-cellular AMP (adenosine monophosphate) concentration, as AMP is cleaved to produce NH4<sup>+</sup> ions, indispensable for cell growth. This event results in the inactivation of iso-citrate dehydrogenase in the TCA cycle and, consequently, the accumulation of both iso-citric and citric acids into the mitochondria. When the intra-mitochondrial citric acid concentration reaches a critical value, citrate enters the cytoplasm in exchange for malate. Citric acid is then either cleaved by ATP-citrate lyase (ACL), the key enzyme of the lipid accumulation process in oleaginous microorganisms, to yield acetyl-CoA and oxaloacetate, or it is secreted to the culture medium [37,66,68]. For the case of lipid-accumulating microorganisms, the resulting acetyl-CoA is carboxylated by acetyl-CoA carboxylase (ACC1) to form malonyl-CoA, the substrate for the biosynthesis of acyl-CoA esters and, subsequently, triacylglycerols [37,66,68].

*Y. lipolytica* strains are a most untypical example of the group of oleaginous yeasts [68]. Lipid content of various strains during growth on glucose under nitrogen limitation (condition favoring lipid storage) is not very high in several cases (e.g. biomass in DCW ≈ 36% *w*/*w*). It certainly does not compare favorably for lipid accumulation with species of *Rhodotorula*/*Rhodosporidium*, *Lipomyces*, and other genera [66,68]. In some cases observed during growth in a shake-flask or batch bioreactor experiments, growth of *Y. lipolytica* in nitrogen-limited glucose- or glycerol-based media resulted in sequential production of intra-cellular lipid and extra-cellular citric acid. In the first steps of nitrogen limitation, lipid accumulation was triggered (the maximum lipid in DCW was rarely >25%, *w*/*w*), and thereafter, lipid content decreased with time, even though significant substrate quantities remained unconsumed in the medium. The period of intra-cellular lipid degradation (turnover) coincided with the secretion of citric acid in non-negligible quantities into the culture medium [25,28,35,53,63,67]. On the other hand, in other studies, as in the current investigation, lipid content as a proportion of the DCW constantly increased over the whole range of the nitrogen-limited batch fermentation, with simultaneous significant citric acid secretion [62,65]. The above-mentioned complex regulation makes it difficult in many instances to obtain high rates of lipid accumulation in batch culture, since, in such conditions, lipid accumulation and citric acid production occur simultaneously, resulting in only moderate lipid accumulation, as in the current investigation. Potentially, this fact was the reason for which, in the first reports that have appeared, the ability of yeasts of the species *Y. lipolytica* to produce lipids from glucose, glycerol, or similarly metabolized compounds has been characterized as "dubious" [53,66,68].

In the trials performed by *Y. lipolytica* ACA-YC 5031, the produced fatty acids (FAs) were palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), and linoleic (C18:2) acid. Oleic acid being the dominant fatty acid under all circumstances, is in agreemen<sup>t</sup> with previous reports [4,20,25,27,42,58,62], indicating that SCO produced by *Y. lipolytica* ACA YC-5031 through the type of the conversion proposed, can constitute perfect precursors for the synthesis of second-generation biodiesel [20,50]. Fatty acid desaturase activity during cultivation was estimated by calculating the ratios of desaturase product to substrate (C16:1/C16:0; C18:1/C18:0; C18:2/C18:1). High C18:1/C18:0 ratios can be observed, in accordance with previous studies carried out by several wild-type mutant *Y. lipolytica* strains tested [4,20,27,42,43], indicating an important Δ9-desaturase activity in the yeas<sup>t</sup> cells, as reported by Rymowicz et al. [27] and Sarris et al. [4]. Sarris et al. [62] found that the addition of OMW in the culture medium increased the amount of oleic acid over 60.0% in time, while it reduced that of C18:0. However, in this study, the addition of OMW slightly reduced the amount of oleic acid, while the addition of increased salt concentration resulted in a slightly further reduction of the above-mentioned cellular FA (see Table 3). Nevertheless, cellular C18:0 concentrations were always ≈60.0% *<sup>w</sup>*/*<sup>w</sup>*, showing that addition of salt to the OMW/glycerol blends affected to the minimum the FA composition of the cellular lipids produced (Table 3).

In all trials performed, and irrespective of the addition of NaCl into the OMW-based media employed, *Y. lipolytica* ACA-YC 5031 performed a significant removal of phenolic compounds into the medium (between 40.0–62.0% *w*/*w*), while a non-negligible decolorization was also observed (Figure 4a,b). At the highest NaCl concentrations employed, a lower decolorization rate was observed, but, in any case and in accordance with the literature [3,7,8,11,62], the removal of phenolic compounds and the removal of color from the fermented OMW-based media was not proportional, meaning, in fact, that high decolorization did not mean that simultaneously high removal of phenolic compounds occurred, and vice versa. Sarris et al. [3] reported 55.9% decolorization and simultaneous 12.9% removal of phenolic compounds when somehow low initial concentrations of phenolic compounds were employed (≈1.80 g/L), while the respective values of removal of phenolic compounds and color were 50.9% *w*/*w* and 45.3% when OMW-based media with initial phenolic compounds ≈5.50 g/<sup>L</sup> were employed for the related strain *Y. lipolytica* ACA-YC 5033 in OMW and glucose-enriched media. Similarly, the yeas<sup>t</sup> *Y. lipolytica* ACA-YC 5029, as reported by Sarris et al. [4], performed up to 10.0% *w*/*w* phenols removal and up to 30.0% color removal during growth on crude glycerol. In general, the ability of some yeasts and fugal strains to remove phenolic compounds depends on the secretion of extracellular oxidases, laccases, and other enzymes, such as lignin peroxidases and manganese-dependent (or independent) peroxidases [4,8,11]. However, the yeasts do not have the appropriate mechanisms to produce those enzymes and to remove the phenolic compounds. Therefore, the phenol and color reduction might be attributed to potential adsorption of phenolic compounds in the yeas<sup>t</sup> cells or even to their partial utilization as carbon source and energy [4].

### **4. Materials and Methods**

### *4.1. Microorganism, Media, and Culture Conditions*

The yeas<sup>t</sup> *Y. lipolytica* ACA-YC 5031 isolated from various types of sourdoughs [69] was kindly offered by the Laboratory of Dairy Science, Agricultural University of Athens, Greece. The organism was maintained on YPDA slants (10 g/<sup>L</sup> glucose, 10 g/<sup>L</sup> yeas<sup>t</sup> extract, 10 g/<sup>L</sup> peptone, and 20 g/<sup>L</sup> agar) at 4 ◦C. The experiments were carried out in 250-mL Erlenmeyer flasks, containing 50 ± 1 mL of growth medium, sterilized at *T* = 121 ◦C for 20 min and inoculated with 1 mL of 24-h exponential pre-culture yeas<sup>t</sup> incubated at 3.0 Hz at 28 ± 1 ◦C. The yeas<sup>t</sup> pre-culture was carried out in yeas<sup>t</sup> extract-glucose-dextrose medium with 10.0 g/<sup>L</sup> of each. Biodiesel-derived glycerol (initial glycerol concentration—Glol0 = 70 ± 5 g/L) was used as a carbon source in the medium, while peptone and yeas<sup>t</sup> extract, 1.0 g/<sup>L</sup> each, were used as a nitrogen source. Two types of fermentation were examined a) containing OMWs that were added in order to yield in initial concentration 2.0 ± 0.20 g/<sup>L</sup> of phenolic compounds and b) without OMW in the culture medium. The origin and the composition of crude glycerol and the chemical composition of OMWs employed in the current investigation were as in Sarris et al. [4]. Specifically, OMWs were received from a three-phase decanter olive mill located in Chania (Crete, Greece) and were frozen at *T* = –20 ◦C. Prior to the experiments, OMWs were de-frozen, and the solids were removed by centrifugation (9000 × g/15 min at *T* = 21 ◦C) in a Universal 320R-Hettich centrifuge (Tuttlingen, Germany). OMW phenolic content expressed as gallic acid equivalent was ~3.5 g/L, while no reducing sugars were found into the wastewaters. Moreover, negligible quantities of olive oil (*c.* 0.2 g/L; determination of oil conducted after triple extraction with hexane) were presented into the OMWs tested. Therefore, in the trials performed, the sole carbon source employed was crude glycerol. The culture medium salt composition was (g/L): KH2PO4,7.0; Na2HPO4×2H2O, 2.5; MgSO4×7H2O, 1.5; MnSO4×H2O, 0.06; ZnSO4×7H2O, 0.02; FeCl3×6H2O, 0.15; CaCl2×2H2O, 0.15 [38]. As a nitrogen source, peptone and yeas<sup>t</sup> extract were used in a concentration of 1.0 g/<sup>L</sup> each, imposing nitrogen-limited conditions in all trials. Experiments were carried out evaluating the effect of NaCl at different concentrations (0.0%, 1.0%, 3.0%, and 5.0% *w*/*v*) in the culture medium. All fermentations carried out in shake-flask mode, and flasks were placed in an orbital shaker (New Brunswick Sc, USA) at an agitation rate of 3.0 Hz (T = 28 ± 1 ◦C). Initial pH in the culture media was adjusted to 6.0 while pH value during the trials was measured and when necessary it was adjusted between the range 4.8–5.8 by adding (periodically and aseptically) small quantities NaOH 5M (e.g. 500–600 μl) [3,11]. The exact volume of NaOH solution needed for the pH correction was evaluated by measuring the volume of NaOH solution required for pH correction in one (at least) flask (collected daily). Then the appropriate volume of NaOH solution was aseptically added in the remaining flasks and the value of pH reached was verified to be in the range of 4.8–5.8.

### *4.2. Dry Weight Determination*

The whole content of the 250 mL flasks was collected, and cells were harvested by centrifugation at 9000 × g/10 min at *T* = 4 ◦C using a Universal 320R-Hettich centrifuge (Tuttlingen, Germany). The pellet was then washed with distilled water, and centrifugation was applied one more time under the same conditions. Biomass (*X*) was dried at *T* = 90 ± 5 ◦C for 24 hours to obtain the dry cell weight (DCW) expressed in g/<sup>L</sup> [38]. Biomass yield *YX*/*Glol* (g/g), was expressed as the grams of cell dry weight (*X*) produced, per grams of substrate (glycerol; *Glol*) consumed (g DCW / g glycerol consumed).

### *4.3. Determination of Total Intra-Cellular Polysaccharides (IPS)*

Determination of IPS was carried out using a modified protocol described by Liang et al. [70] and the 3,5-dinitrosalicylic acid method (DNS) described by Miller [71]. Specifically, 0.05 g DCW were acidified by adding 10 mL HCl 2M. The solution was then hydrolyzed at *T* = 100 ◦C for 30 min, followed by addition of 10 mL NaOH 2M to reach pH = 7.0, and was subsequently filtered through Whatman filter paper twice. Then, 0.5 mL of the sample solution and 0.5 mL DNS reagen<sup>t</sup> were

transferred into tubes and left in a water-bath at *T* = 100 ◦C for 5 min, followed by 2 min a *T* = 25 ◦C. Finally, 5 mL distilled water were added to the samples mixing well, and the absorbance at 540 nm was measured using a Hitachi U-2000 Spectrophotometer (Tokyo, Japan).

### *4.4. Determination of Glycerol, Polyols, and Citric Acid*

The concentration of the remaining glycerol, the produced polyols, and citric acid were determined during the fermentation using Waters 600E High-performance liquid chromatography (HPLC), (Waters Association, Milford, MA, USA) [72]. The samples were first filtered using a membrane of 0.2 μm diameter, and 10 μl of the sample were injected. The mobile phase was H2SO4 5 mM, while the static phase was the column Amimex HPX-87H (Biorad, Richmond, CA, USA) (30 cm x 7.8 mm). The column flow was 0.5 mL/min, at *T* = 45 ◦C. The apparatus type was Waters 600E with RI detector (RI; Waters 410) for the determination of glycerol and polyols and UV detector (Waters 486) for the determination of organic acids. The area of each compound was determined according its retention time, and the concentration of each compound (glycerol, citric acid, mannitol, erythritol) was determined using reference curves and expressed as g/L. Citric and iso-citric acid were not totally separated with the implicated HPLC analysis method, and the reported concentration corresponds to the sum of these acids, expressed as total citric acid (*Cit*). In order to proceed with a more precise determination of iso-citric acid, in some of the fermentation points, an enzymatic method, based on the measurement of the NADPH2 produced during conversion of the iso-citric to α-ketoglutaric acid, the reaction catalyzed by the iso-citrate dehydrogenase, was employed. In all points where iso-citrate determination occurred, iso-citric acid represented a quantity of 5–7% ( *w*/*w*) of total citric acid produced, regardless of the culture conditions employed.

Glycerol assimilation rate (in g/L·h) was expressed as substrate removed (g) per L of medium and per hour (= −ΔGlol/Δt) for the respective time in which fermentation was performed.

Total citric acid yield, *YCit*/*Glol* (g/g), was calculated based on the grams of produced total citric acid (*Cit*) per grams of substrate consumed (*Glol*).

Citric acid volumetric productivity (in g/L·h) was determined as the concentration of citric acid in a given fermentation time divided by this respective time.

The global yield of the produced citric acid per glycerol consumed ( *YCit*/*Glol*) was determined as the produced citric acid in a plot against the consumed substrate [Cit = *f* (Glol consumed)] with equation y = *a*x + b, where "*a*" represents the citric acid yield *YCit*/*Glol*.

Mannitol yield *YMan*/*Glol* (g/g), was calculated based on the grams of produced mannitol (*Man*) per grams of substrate consumed (*Glol*).

Erythritol yield, *YEry*/*Glol* (g/g), was calculated based on the grams of produced erythritol (*Ery*) per grams of substrate consumed (*Glol*).

### *4.5. Quantitative Determination of the Cellular Lipid and Fatty Acid (FA) Composition Analysis*

Total cellular lipid was extracted from DCW with a chloroform-methanol mixture (30 mL, 2:1, v/v) after 72 h in the darkness [38]. After three days, cell debris were removed through filtration (Whatman ®filter n◦ 3) and the solvent mixture was completely evaporated in a rotary evaporator (R-144, Büchi Labortechnik, Flawil, Switzerland) at *T* = 60–65 ◦C. Total intra-cellular lipid, L, was determined gravimetrically and was expressed as g/L. Lipid in DCW (%, *w*/*w*; *YL*/*X*) was calculated based on percentage of the accumulated lipid (*L*) per produced dry biomass ( *X*).

The extracted intra-cellular lipids were converted to their fatty acid methyl-esters (FAMEs) and analyzed in a gas chromatography (GC) (Fisons 8000 series, O ffenbach, Germany) equipped with an FID (Fisons) according to the method described by Zikou et al. [73]. Methyl-esterification was performed in two phases. One to two boiling stones and 10 mL sodium methoxide (MeO-Na+) were added into the extracted total lipids. The samples were left to boil for 20 min. Methanol hydrochloride (CH3OH-HCl) was then added until the decolorization of the mixture, which reached a milky color. Boiling was then continued for another 20 min before the addition of distilled water to end the reaction. Finally, 6 mL of hexane were added, and the mixture was shaken vigorously. The solvent phase was collected, and a 1 μl sample was injected into GC.

### *4.6. Phenolic Compounds Determination*

Determination of total phenol compounds (pHØ) into the medium was carried out according to the method described by Aggelis et al. [8]. Sampleof 0.2 mL was mixed with 10.8 mL distilled water, 8 mL Na2CO3 (75 g/L), and 1 mL Folin-Ciocalteu reagent. Blank samples used as controls were prepared using 0.2 mL of distilled water. The mixture was shaken and remained in darkness for 2 h. The absorbance was measured at 750 nm in a Hitachi U-2000 Spectrophotometer (Tokyo, Japan). The concentration of phenolic compounds was expressed in equivalence of gallic acid according to a reference curve.
